The present invention relates a DNA polymerase which is useful as a reagent for genetic engineering, a method for producing the same, and a gene encoding thereof.
DNA polymerases are useful enzymes as reagents for genetic engineering, and the DNA polymerases are widely used for method of determining base sequences of DNA, labeling, methods of site-directed mutagenesis, and the like. Also, thermostable DNA polymerases have recently been remarked with the development of the polymerase chain reaction (PCR) method, and various DNA polymerases suitable for the PCR method have been developed and commercialized.
Presently known DNA polymerases can be roughly classified into four families according to amino acid sequence homologies, among which family A (pol I type enzymes) and family B (xcex1 type enzymes) account for the great majority. Although DNA polymerases belonging to each family generally possess mutually similar biochemical properties, detailed comparison reveals that individual DNA polymerase enzymes differ from each other in terms of substrate specificity, substrate analog incorporation, degree and rate for primer extension, mode of DNA synthesis, association of exonuclease activity, optimum reaction conditions of temperature, pH and the like, and sensitivity to inhibitors. Thus, those possessing especially suitable properties for the respective experimental procedures have been selectively used of all available DNA polymerases.
An object of the present invention is to provide a novel DNA polymerase not belonging to any of the above families, and possessing biochemical properties not owned by any of the existing DNA polymerases. For example, primer extension activity and 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity are considered as mutually opposite properties, and none of the existing DNA polymerase enzymes with strong primer extension activity possess 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity, which is an important proofreading function for DNA synthesis accuracy. Also, the existing DNA polymerases possessing excellent proofreading functions are poor in primer extension activity. Therefore, development of a DNA polymerase possessing both potent primer extension activity and potent 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity would significantly contribute to DNA synthesis in vitro.
Another object of the present invention is to provide a method for producing the novel DNA polymerase mentioned above.
A still another object of the present invention is to provide a gene encoding the DNA polymerase of the present invention.
As a result of extensive investigation, the present inventors have found genes of the novel DNA polymerase from hyperthermophilic arcaebacterium Pyrrococcus furious, followed by cloning of the above genes, and have clarified that two kinds of novel proteins possess a novel DNA polymerase activity exhibiting the activity under coexistence of the above two kinds of proteins. Furthermore, the present inventors have prepared a transformant into which the above genes are introduced, and have succeeded in mass-producing the complex type DNA polymerase.
Accordingly, the gist of the present invention is as follows:
[1] A DNA polymerase characterized in that the DNA polymerase possesses the following properties:
1) exhibiting higher polymerase activity when assayed by using as a substrate a complex resulting from primer annealing to a single stranded template DNA, as compared to the case where an activated DNA is used as a substrate;
2) possessing a 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity;
3) being capable of amplifying a DNA fragment of about 20 kbp, in the case where polymerase chain reaction (PCR) is carried out using xcex-DNA as a template under the following conditions:
PCR Conditions:
(a) a composition of reaction mixture: containing 10 mM Tris-HCl (pH 9.2), 3.5 mM MgCl2, 75 mM KCl, 400 xcexcM each of dATP, dCTP, dGTP and dTTP, 0.01% bovine serum albumin, 0.1% Triton X-100, 5.0 ng/50 xcexcl xcex-DNA, 10 pmole/50 xcexcl primer xcex1 (SEQ ID NO:8 in Sequence Listing), primer xcex11 (SEQ ID NO:9 in Sequence Listing), and 3.7 units/50 xcexcl DNA polymerase;
(b) reaction conditions: carrying out a 30-cycle PCR, wherein one cycle is defined as at 98xc2x0 C. for 10 seconds and at 68xc2x0 C. for 10 minutes;
[2] The DNA polymerase according to the above item [1], characterized in that the DNA polymerase exhibits a lower error rate in DNA synthesis as compared to Taq DNA polymerase;
[3] The DNA polymerase according to the above item [1] or [2], wherein the molecular weight as determined by gel filtration method is about 220 kDa or about 385 kDa;
[4] The DNA polymerase according to any one of the above items [1] to [3], characterized in that the DNA polymerase exhibits an activity under coexistence of two kinds of DNA polymerase-constituting protein, a first DNA polymerase-constituting protein and a second DNA polymerase-constituting protein;
[5] The DNA polymerase according to the above item [4], characterized in that the molecular weights of the first DNA polymerase-constituting protein and the second DNA polymerase-constituting protein are about 90,000 Da and about 140,000 Da as determined by SDS-PAGE, respectively;
[6] The DNA polymerase according to the above item [4] or [5], characterized in that the first DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above item [4] or [5] comprises the amino acid sequence as shown by SEQ ID NO:2 in Sequence Listing, or is a functional equivalent thereof possessing substantially the same activity which results from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence;
[7] The DNA polymerase according to the above item [4] or [5], characterized in that the second DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above item [4] or [5] comprises the amino acid sequence as shown by SEQ ID NO:4 in Sequence Listing, or is a functional equivalent thereof possessing substantially the same activity which results from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence;
[8] The DNA polymerase according to item [4] or [5], characterized in that the first DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above item [4] or [5] comprises the amino acid sequence as shown by SEQ ID NO:2 in Sequence Listing, or is a functional equivalent thereof possessing substantially the same activity which results from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence, and that the second DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above item [4] or [5] comprises the amino acid sequence as shown by SEQ ID NO:4 in Sequence Listing, or is a functional equivalent thereof possessing substantially the same activity which results from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence;
[9] A first DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above item [4] or [5], wherein the first DNA polymerase-constituting protein comprises the amino acid sequence as shown by SEQ ID NO:2, or an amino acid sequence resulting from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence, as a functional equivalent thereof possessing substantially the same activity;
[10] A second DNA polymerase-constituting protein which constitutes the DNA polymerase according to the above [4] or [5], wherein the second DNA polymerase-constituting protein comprises the amino acid sequence as shown by SEQ ID NO:4, or an amino acid sequence resulting from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence as a functional equivalent thereof possessing substantially the same activity;
[11] A DNA containing a base sequence encoding the first DNA polymerase-constituting protein according to the above item [9], characterized in that the DNA comprises an entire sequence of a base sequence encoding the amino acid sequence as shown by SEQ ID NO:2 in Sequence Listing, or a partial sequence thereof, or that the DNA encodes a protein having an amino acid sequence resulting from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence of SEQ ID NO:2 and possessing a function as the first DNA polymerase-constituting protein;
[12] A DNA containing a base sequence encoding the first DNA polymerase-constituting protein according to the above items [9], characterized in that the DNA comprises an entire sequence of the base sequence as shown by SEQ ID NO:1 in Sequence Listing or a partial sequence thereof, or that the DNA comprises a base sequence capable of hybridizing thereto under stringent conditions;
[13] A DNA containing a base sequence encoding the second DNA polymerase-constituting protein according to the above item [10], characterized in that the DNA comprises an entire sequence of a base sequence encoding the amino acid sequence as shown by SEQ ID NO:4, or a partial sequence thereof, or that the DNA encodes a protein having an amino acid sequence resulting from deletion, insertion, addition or substitution of one or more amino acids in the amino acid sequence of SEQ ID NO:4 and possessing a function as the second DNA polymerase-constituting protein;
[14] A DNA containing a base sequence encoding the second DNA polymerase-constituting protein according to the item [10], characterized in that the DNA comprises an entire sequence of the base sequence as shown by SEQ ID NO:4 in Sequence Listing or a partial sequence thereof, or that the DNA comprises a base sequence capable of hybridizing thereto under stringent conditions;
[15] A method for producing a DNA polymerase, characterized in that the method comprises culturing a transformant containing both gene encoding the first DNA polymerase-constituting protein according to the above item [11] or [12], and gene encoding the second DNA polymerase-constituting protein according to the above item [13] or [14], and collecting the DNA polymerase from the resulting culture; and
[16] A method for producing a DNA polymerase, characterized in that the method comprises culturing a transformant containing gene encoding the first DNA polymerase-constituting protein according to the above item [11] or [12], and a transformant containing gene encoding the second DNA polymerase-constituting protein according to the above item [13] or [14], separately; mixing DNA polymerase-constituting proteins contained in the resulting culture; and collecting the DNA polymerase.